Cell death inducers for mast cells

ABSTRACT

A cell death inducer for mast cells, a preventive/therapeutic agent for diseases in which mast cells are implicated, having a fusion protein including a PTD and an MITF variant as an active ingredient and a fusion protein comprises a His Tag, a PTD, and an MITF variant, wherein the MITF variant being an MITF mi variant, wh variant, HLH fragment, or A-type N-terminal region (1-305) fragment, and the PTD being a TAT-derived peptide; DNA coding for the fusion protein; and a method for preparing the fusion protein using genetic engineering techniques. Actions of the fusion protein of the present invention include inhibiting an activity of endogenous MITF by translocating into mast cells, inhibiting a survival of mast cells derived from precursor cells, and inducing cell death (apoptosis) in mature mast cells.

[0001] This application claims priority from Japanese Patent Application No. 2001-204567, which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to medical use of MITF variants. More specifically, it relates to medical use of fusion proteins having the MITF variant as a component.

BACKGROUND

[0003] MITF (an abbreviation for microphthalmia-associated transcription factor) is one of transcription regulators present in organisms; it is a protein capable of modulating an expression of a c-kit gene, which is specific to mast cells.

[0004] MITF is a known substance (Cell, vol. 74, 395404, 1993); it was, however, a gene that codes for MITF that was first discovered. That is to say, the gene was isolated as a causal gene for a mi/mi mouse. In the mi/mi mouse, because of a mutation in the MITF gene, that is, a deletion of one amino acid in the transcription activation region of the MITF gene, normal MITF is not expressed. The mi/mi mouse is a mutant mouse having hypoplasia of an eyeball, melanocyte deficiency, mast cell loss, and osteopetrosis of a bone as main symptoms thereof, and presents differentiation anomalies in tissues, such as melanocytes, mast cells, retinal pigment epithelial cells, and osteoclasts. These cell differentiation disorders in the mi/mi mouse are attributed to the fact that normal MITF is not expressed, and gene transcription is not activated by MITF.

[0005] Furthermore, in Northern-blot analyses of a tissue distribution of an expression, using tissues or cell lines from a normal mouse, it was found that MITF mRNA was expressed in the heart, in melanocytes, and in mast cells. In recent years, the existence of MITF isoforms has been reported; i.e. a melanocyte type (M type), a heart type (H type), and an A-type, in which the cDNA sequences were different at the 5′ end [Seikagaku (Journal of the Japanese Biochemical Society), vol. 71, no. 1, 61-64, 1999]. The MITF gene comprises 10 (M type) or 11 (A and H types) exons, and all three types are substantially common beyond exon 2. In the M type, two further types are distinguished by an addition, or lack thereof, of exon Sb, which comprises 18 bases coding for 6 amino acids. The A and H types completely match each other beyond exon 1B, exon 1 at the 5′ end being different. The A and H types and the M type are common beyond exon 2; however the M type has no exon 1 B upstream of exon 2, which is linked to a specific exon 1. Furthermore, it has been shown that the promoter for each type is different from the genomic sequence.

[0006] An MITF protein is assumed to comprise a nuclear localization region, a transcription activation region, a DNA binding region, a dimerization region, and an activation region for MITF itself; the presence of these regions is common to all known types. The MITF protein is a transcription regulator, which has a bHLH-Zip (base-helix-loop-helix/leucine zipper) motif at a center of its structure, it forms a dimer to bind to DNA, and activates transcription of targeted genes. Since major differences have been reported between transcription activation capabilities of the A-type and the H type, there is assumed to be a function that regulates the transcriptional activity at exon 1, which differs in genetic sequence between the two types.

[0007] Furthermore, it has been reported that the MITF protein works as a transcription regulator in melanocytes, and that it is involved in the multiplication and differentiation of melanocytes, as well as in the melanin synthesis pathway, and the like.

[0008] It has been reported that the MITF protein also acts as a transcription regulator in mast cells, modulating the expression of the c-kit gene (a transcription factor that activates the c-kit promoter) (Blood, vol. 88, no. 4, 1225-33, 1996). The c-kit gene is expressed in hematopoietic precursor cells, mast cells, pigment cells, and germ cells and regulates the multiplication and the differentiation of these cells by the action of the Si factor. It is thought that, in mast cells, MITF protein is involved in mast cell survival maintenance by regulating the expression of c-kit gene expression.

[0009] Mast cells have long been reported to be involved in allergic diseases [“IgE, Mast Cells and the Allergic Response” (Ciba Foundation symposium) 147, John Wiley & Sons, Chichester, UK, 1989]. Furthermore, diseases other than allergic diseases in which mast cells are involved include autoimmune diseases, pulmonary fibrosis, carcinomas, mastocytosis, mastocytoma, and the like.

[0010] Meanwhile, PTD (an abbreviation for Protein Transduction Domain) is a general term for domains to penetrate the biological membrane and transfer proteins into the cells (uptake). For example, in analysis of HIV antigens by domain units, it is confirmed that a TAT-derived peptide portion serves to transfer the HIV antigen inside normal T-cells, which is one of contributing factors in cell infection (Cell, vol. 55, 1179-88, 1988). Based on such observations, there have been reports of the existence of various PTDs to work in a similar way to TAT, and of techniques whereby these PTDs are fused with various proteins for translocation into cells (Current Opinion in Molecular Therapeutics 2000, vol. 2, no. 2, 162-67, 2000).

[0011] However, there have been no reports to date of techniques related to MITF or mast cells to transfer into cells using PTD.

DISCLOSURE OF THE INVENTION

[0012] An object of the present invention is to provide a novel medical use for MITF variants.

[0013] As a result of studies undertaken by the present inventors in view of the situation described above, it was discovered that, by combining MITF variants and PTD, it is possible to induce cell death in mast cells, and thus the present invention was completed.

[0014] That is to say, one aspect of the present invention is a cell death inducer for mast cells, having a fusion protein comprising a PTD and an MITF variant as an active ingredient.

[0015] One aspect of the present invention is a preventive/therapeutic agent for diseases in which mast cells are implicated, having a fusion protein comprising the PTD and the MITF variant as the active ingredient.

[0016] One aspect of the present invention is a fusion protein wherein this fusion protein comprises a His Tag, the PTD, and the MITF variant, the MITF variant being an MITF mi variant, wh variant, HLH fragment, or A-type N-terminal region (1-305) fragment, and the PTD being a TAT-derived peptide.

[0017] One aspect of the present invention is a pharmaceutical composition comprising the fusion protein and a pharmacologically acceptable carrier.

[0018] One aspect of the present invention is a method for preparing the fusion protein, the method comprising a step of producing the fusion protein using genetic engineering techniques.

[0019] One aspect of the present invention is DNA that codes for the fusion protein.

[0020] One aspect of the present invention is a method for inducing cell death in mast cells comprising administrating an effective dose of a fusion protein comprising a PTD and an MITF variant.

[0021] One aspect of the present invention is use of a fusion protein comprising a PTD and an MITF variant for preparing an agent for inducing cell death in mast cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 schematically illustrates the structures of fusion proteins of the present invention (mi form and wh form) and plasmids pSU082 (mi form/pTrcHisB) and pSU083 (wh form/TrcHisB) that express fusion proteins.

[0023]FIG. 2 schematically illustrates the structure of a fusion protein of the present invention (HLH form) and a plasmid pSU085 (HLH form/pTrcHisB) that expresses the fusion protein.

[0024]FIG. 3 schematically illustrates a procedure for constructing a plasmid pSU087 (A-type N-terminal region/pSU093) that expresses a fusion protein of the present invention (A-type N-terminal region).

[0025]FIG. 4 schematically illustrates a procedure for constructing a plasmid pSU121 (wh form/pET14) that expresses a fusion protein of the present invention (wh form).

DETAILED DESCRIPTION

[0026] Modes of embodiment for the present invention will be described in further detail hereinafter. The detailed description below is illustrative, being solely explanatory in intent, and does not limit the present invention in any way.

[0027] Furthermore, the technical and scientific terms used in the present invention, unless defined separately, have meanings that are normally understood by a person of ordinary skill in the technical field to which the present invention belongs. In the present invention, various methods known to those skilled in the art are referenced. The content of publications, and the like, which disclose such cited well-known methods, are incorporated herein by reference, as if completely set forth in their entirety in the present specification.

[0028] A. MITF Variant

[0029] The MITF variant used in the present invention is no particular restriction thereon as long as it is a natural type (wild type) MITF variant, and it has an MITF inhibitory activity. Specifically, examples thereof include the mi variant of MITF, the wh variant of MITF, and other MITF variants, for example, splicing variants (which may be derived from any of the A, H, M, or N isoforms), or a partial MITF structure having an MITF inhibitory activity, for example, a bHLH-Zip (the N-terminal residues 196 to 285 of the M isoform) fragment, an N-terminal end fragment comprising this portion, an N-terminal region of the A isoform (from the N-terminal residue 1 to 305) fragment, and the like. Specifically, examples include the MITF variant disclosed in Trends in Genetics, vol. 11, no. 11, 44248, 1995; WO 00/47765; WO 01/66735 (Japanese Patent Application No. 2000-63959), and the like. Particularly preferred variants are the mi variant, the wh variant, the bHLH-Zip fragment (hereinafter, HLH fragment), and the N-terminal region (1-305) fragment of the A isoform. The relationships between the amino acid sequences thereof and base sequences are as shown in Table 1. TABLE 1 Amino Acid Preferable Base MITF Variant Sequence Sequence mi variant SEQ ID NO: 1 SEQ ID NO: 2 wh variant SEQ ID NO: 3 SEQ ID NO: 4 HLH fragment SEQ ID NO: 5 SEQ ID NO: 6 A-type N-terminal SEQ ID NO: 21 SEQ ID NO: 22 region

[0030] Furthermore, the scope of the present invention includes structural analogs of these variants having substantially the same degree of MITF inhibitory activity as these variants. For example, these may be variants wherein one or a plurality of amino acids are substituted, deleted, inserted, or added in the above-mentioned amino acid sequences, and which have substantially the same degree of activity as these variants.

[0031] B. PTD

[0032] There is no particular restriction on the PTD used in the present invention, so long as it has the property of allowing uptake (transfer) into cells, and well-known PTDs may be used. Specifically, examples include the various oligopeptides listed in Table 2 on page 164 of the above-mentioned reference (Current Opinion in Molecular Therapeutics 2000, vol. 2, no. 2, 162-67, 2000), the various PTD oligopeptides disclosed in WO 99/10376, WO 99/29721, JP-05-505102-T, and the like.

[0033] A preferable PTD is a TAT-derived peptide (the amino acid sequence is indicated by YGRKKRRQRRR, SEQ ID NO: 7, and the preferred base sequence is indicated by SEQ ID NO: 8).

[0034] The PTD may be linked to either the N-terminal end or the C-terminal end of the MITF variant. Furthermore, the linkage may be either direct, or indirect via a cross-linking agent (linker). Examples of linkers include glycine residues, and the like.

[0035] C. Other Components (Domains)

[0036] The fusion protein of the present invention may be linked to a domain that has affinity to a ligand in affinity chromatography for purification. There are no particular restrictions on the domain, and well-known domains may be used. In this regard, examples include an antigen and an antibody, a receptor and a ligand, Ni-NTA (nitrilotriacetic acid) and a His Tag, avidin (or streptavidin) and biobn, and the like. A His Tag (the amino acid sequence is represented by MGGSHHHHHH, SEQ ID NO: 9, the preferred base sequence is represented by SEQ ID NO: 10) is preferred.

[0037] This domain may be linked to either the N-terminal end or the C-terminal end of the fusion protein. Furthermore, the linkage may be either direct, or indirect via a cross-linking agent (linker). Examples of linkers include glycine residues, and the like.

[0038] D. Preparation of the Fusion Protein

[0039] Examples of methods for preparing the fusion protein of the present invention include: (1) a method for synthesizing the entire fusion protein using chemical synthesis techniques; (2) a method wherein each component (domain) mentioned above is synthesized separately and then linked using chemical reaction means; and, (3) a method wherein the genes coding for each component are linked and are then all expressed as the fusion protein at one time using genetic engineering techniques, and the like. In the case of (2), methods for preparing each component include chemical synthesis methods, cell culture methods, methods using genetic engineering techniques, and the like. A PTD may also be prepared by cleavage and isolation from an HIV antigen.

[0040] A case will be described by way of example wherein the fusion protein is prepared by genetic engineering techniques.

[0041] 1) DNA coding for the fusion protein is prepared. The preparation uses conventional methods. First, the gene coding for the MITF variant is prepared. The gene can be prepared by extracting mRNA from suitable cells, synthesizing cDNA using reverse transcriptase and DNA polymerase, and amplifying this with the polymerase chain reaction (PCR) method. Specifically, mRNA can be extracted using commercially available mRNA extraction kits, and the like; reverse transcription, cDNA synthesis, and amplification may be carried out by the 5′-RACE method using commercially available cDNA amplification kits, and the like (Proc. Natl. Acad. Sci. USA, vol. 85, 8998-9002, 1988), or by the reverse transcription polymerase chain reaction (RT-PCR) method, and the like, using suitable primers. Furthermore, the gene can also be obtained by extracting genomic DNA from a suitable cell and amplifying by PCR. Furthermore, the gene coding for MITF disclosed in well-known publications, such as the aforementioned Cell (1993) and WO 01/66735, may also be used. For example, the HLH fragment of the present invention may be prepared from an M-type MITF, and the N-terminal region (1-305) fragment of the A-type from an A-type MITF (MITF A/pCDNA3), respectively.

[0042] Next, a gene coding for PTD is prepared by the same method as described above and ligated to the gene coding for the MITF variant. The His Tag is prepared in the same way. Furthermore, the DNA coding for the fusion protein of the present invention can be prepared by inserting DNA coding for a fusion protein comprising a PTD-MITF variant, using a commercially available plasmid bearing the His Tag (pTrcHisB, Invitrogen).

[0043] 2) An expression vector is prepared by integrating DNA into a suitable vector. The vector is used to express the fusion protein under the control of a specific promoter. This integration is performed by conventional methods.

[0044] It is possible to construct a host/vector system by purifying the target DNA obtained as described above and inserting it into vector DNA. A host combined with a replicon derived from species that is compatible with a host cell is generally used for the host/vector system. The vector DNA has an origin of replication, a promoter, a regulatory sequence (enhancer), a signal sequence, a ribosomal binding site, an RNA splicing sequence, a polyA addition site, a transcription termination sequence (terminator), and the like. Furthermore, it may have a marker sequence that allows phenotypic selection among transformed cells. Examples of the vector DNA include: vectors derived from chromosome and episome, vectors derived from bacterial plasmid, bacteriophage, vectors derived from virus such as baculovirus, papovavirus and SV40, cosmid, phagemid, and the like. Furthermore, an expression vector, a cloning vector, and the like, may be used depending on the purpose.

[0045] The promoter includes well-known promoters and can be selected according to the host for expression. For example, in cases where Escherichia coli is the host, examples include promoters such as a trp promoter, a lac promoter, a trc promoter (a synthetic promoter in which a −35 region of the trp promoter and a −10 region of the lac promoter are ligated), and the T7 promoter. Furthermore, the expression vector may bear a marker gene, such as amp^(r).

[0046] Methods well known per se may be used as the method for integrating the DNA according to the present invention into the vector DNA. For example, a method may be used wherein a suitable restriction endonuclease is selected and applied to cleave the target DNA at specific sites, which is then mixed with a similarly treated vector DNA and re-linked by a ligase. Alternately, the intended recombinant vector can also be obtained by ligating a suitable linker with the target DNA and inserting this into the multicloning site of the vector that is appropriate for the purpose. Furthermore, the target protein can be prepared by using the expression vector as the vector DNA which is introduced into a host.

[0047] 3) Transformants are prepared by introducing the expression vector into hosts. The transformations are performed by conventional methods. Escherichia coli, Bacillus subtilis, yeast, animal cells, and the like, may be used as the host. Escherichia coli is preferred. Furthermore, auxotrophic strains and antibiotic-sensitive strains may also be used as hosts.

[0048] Methods for preparing the transformants include methods for introducing a plasmid directly into host cells, methods for integrating the plasmid into the chromosome, and the like. The former includes the protoplast polyethylene glycol method, the electroporation method, and the like. For the latter, a portion of the DNA sequence of a gene that is present in a host chromosome may be included in a plasmid, and using a homologous sequence portion, the plasmid, or a linear fragment, is integrated into the host chromosome by homologous recombination.

[0049] 4) Transformants are cultured to produce the fusion protein. Culture is performed by conventional methods, using a suitable culture medium and suitable culture conditions (temperature, time, etc.) depending on the host. In the case where Escherichia coli is used, in general, this is performed under culture conditions of approximately 1 543° C. (preferably 30-37° C.) and approximately 1-100 hours. Furthermore, aeration and agitation can also be added as necessary. The culturing system may be any of batch culture, semi-batch culture (fed-batch culture), or continuous culture.

[0050] 5) The fusion protein produced is purified. In the case where Escherichia coli is used as the host, the protein is first solubilized by treatment such as sonication of cells. Purification of the fusion protein produced may be performed by techniques well known perse (WO 99/55899, and the like). Examples include methods using an Ni column, anion exchange treatments, dialysis treatments, and the like. Furthermore, use of the fusion protein of the present invention, obtained by treatment with a denaturing agent (chaotropic agent) and subsequent removal of the denaturing agent, is preferred. Examples of denaturing agents include urea, guanidine hydrochloride, thiocyanate, and the like. Conditions for adding the denaturing agent during treatment with the denaturing agent may, for example, be concentrations of approximately 1-10 M. Specifically, after treatment by contacting the fusion protein and the denaturing agent, a treatment is carried out using the Ni column in the presence of the denaturing agent; furthermore, operations to eliminate the denaturing agent are carried out by anion exchanger or dialysis treatment, so as to purify the fusion protein.

[0051] E. Characteristics of the Prepared Fusion Protein

[0052] The fusion protein of the present invention has a property of transducing into cells, in particular, into mast cells. Preferably, it comprises a His Tag, a PTD, and an MITF variant. It has a molecular weight of approximately 10-100 kilo Daltons (kDa). Preferably, it is laid out in order, from the N-terminal end, as His Tag, PTD, and MITF variant. Specific sequences are as shown TABLE 2 Amino Acid Preferable Base Fusion Protein Sequence Sequence His Tag-PTD-mi variant of MITF SEQ ID NO: 11 SEQ ID NO: 12 His Tag-PTD-wh variant of MITF SEQ ID NO: 13 SEQ ID NO: 14 His Tag-PTD-fragment of HLH SEQ ID NO: 15 SEQ ID NO: 16 His Tag-PTD-A-Type N-terminal SEQ ID NO: 23 SEQ ID NO: 24 region of MITF

[0053] F. Formulation

[0054] Techniques well known per se can be used to formulate the fusion protein of the present invention. For example, a pharmacologically acceptable carrier can be added to, or mixed with, the fusion protein. Examples of concentrations for the fusion protein in the pharmaceutical composition obtained by formulation include approximately 0.1-100 μg/mL or 0.1-100 nM.

[0055] G. Use application

[0056] The MITF variant of the present invention (or, the fusion protein using the same) has such effects to inhibit the activity of endogenous MITF by translocating inside the mast cell, to inhibit the differentiation of mast cells from precursor cells, to inhibit the survival of mast cells, and to induce cell death (apoptosis) in mature mast cells. Therefore, it is anticipated that the formulation of the present invention will be useful in prevention/treatment of various diseases in which mast cells are implicated. Examples of these diseases include allergies, asthma, autoimmune diseases, pulmonary fibrosis, carcinomas, mastocytosis, mastocytoma, and the like.

[0057] H. Dosage and Administration

[0058] In terms of dosage and administration for the fusion protein of the present invention, the dosage may be selected so that the fusion protein is present at in vivo concentrations of approximately 0.001-10 μg/mL. Alternatively, for example, dosages may be of the order of 10 μg-50 mg. Administration routes include intravenous administration, subcutaneous administration, intramuscular administration, percutaneous administration, tracheobronchial administration, and the like.

EXAMPLES

[0059] Advantages, features, and possibilities for the present invention will be described hereinafter in more detail referring to illustrative examples; the present invention is not, however, limited to the following examples.

Example 1

[0060] 1) Construction of the Expression Plasmid for the Fusion Protein

[0061] The expression plasmid was constructed by inserting a gene for a His Tag-PTD-MITF variant, wherein a PTD sequence is connected downstream of a His Tag for purification, downstream of which the cDNA for the MITF variant is connected, between the NcoI site, which is located upstream of the His Tag sequence, and the HindIII site in the multicloning site of the Escherichia coli expression vector pTrcHisB (Invitrogen, No. V360-20). See FIG. 1. His Tag and the PTD sequences were added to the MITF cDNA by PCR. First, with the cDNA of M type MITF (pSU054, MITF-MpT7 Blue) as the template, the upstream portion that contains MITF and the BamHI site was amplified by PCR. Furthermore, this was elongated using four kinds of primer (M-tat, Tat 3, Tat 2, and Tat 1) until the His Tag and the recognition sequence for the restriction endonuclease NcoI were added upstream of the transcription initiation codon of MITF, then cloned between the NcoI site and the BamHI site of the Escherichia coli expression vector pTrcHisB (this bears the lac P/O and amp^(r)).

[0062] The primers have following base sequences. M-tat (SEQ ID NO: 17): GCGACGAAGAGGTATGCTAGAATACAGTCACTACC Tat 3 (SEQ ID NO: 18): GGCAGGAAGAAGCGGAGACAGCGACGAAGAGGTATG Tat 2 (SEQ ID NO: 19): ATCATCATCATGGTGGTTATGGCAGGAAGAAGCGG Tat 1 (SEQ ID NO: 20): TAAACCATGGGGGGTTCTCATCATCATCATCATCATGGTG

[0063] Next, by digesting the M type (pSU054), the mi variant (pSU061, MITF-M mi/pT7 Blue), and the wh variant (pSU062, MITF -M/pT7 Blue) with BamHI and HindIII, respectively, a downstream portion of the MITF cDNA was isolated. The mutant portion is also contained in this portion. The isolated cDNA fragment was inserted between the BamHI site and the HindIII site of the plasmid into which the upstream portion was cloned. The pSU81 plasmid has inserted therein a fusion protein gene that contains the M type, which is the normal type MITF. pSU082 and pSU083 each have a fusion protein inserted therein, containing the mi variant and the wh variant, respectively.

[0064] 2) Expression of the Fusion Protein

[0065] The constructed expression plasmid was introduced into Escherichia coli DH5a (Toyobo) for transformation. A volume of 50 mL of L-Broth (containing 50 μg/mL ampicillin) was inoculated with 1 platinum loop of Escherichia coli and cultured at 37° C. for 17 hours. Furthermore, 1 L of L-Broth (containing 50 μg/mL ampicillin and 0.2% glucose) was inoculated so as to obtain a 1% culture solution. After culturing at 37° C. until the A₆₀₀ turbidity reached approximately 0.1, IPTG (isopropyl thio-β-D-galactoside) was added so as to obtain a final concentration of 0.4 mM. After culturing for 2 hours, Escherichia coli was harvested.

[0066] 3) Purification of the Fusion Protein

[0067]Escherichia coli was recovered by centrifugation, sonicated in a 20 mM HEPES buffer solution (pH 8.0; hereinafter, pH is the same) that contains 8 M urea, 0.1 M sodium chloride, and 10 mM DTT, and solubilized. The obtained lysate was applied to a Ni-NTA agarose (Qiagen) column, which is equilibrated with a 20 mM HEPES buffer solution containing 8 M urea, 0.1 M sodium chloride, 10 mM imidazole, and 1 mM DTT and washed with the same buffer solution. The protein bound to the column was eluted by the imidazole linear concentration gradient method, using the same buffer solution as the A solution and the same buffer solution, containing 200 mM imidazole, as the B solution, and analyzed by SDS-PAGE (Tefco); the fractions showing the target molecular weight were pooled.

[0068] A solution, diluted by adding both 1 volume of 20 mM HEPES buffer solution and 2 volumes of the same buffer solution containing 4 M urea to 1 volume of the eluate, was applied to a quatemary ammonium strong anion exchanger (product name: Q-Sepharose, Pharmacia), which is equilibrated with a 20 mM HEPES buffer solution containing 4 M urea and 25 mM sodium chloride, and washed with the same buffer solution. Next, after eliminating the urea by washing with a 20 mM HEPES buffer solution, the protein bound to the column was eluted with the same buffer solution containing 1 M sodium chloride. After the eluted fusion protein was dialyzed against Dulbecco's PBS (isotonic phosphate-buffered solution) containing 10% glycerol, it was dispensed and stored at −80° C.

[0069] When a final product that was obtained was analyzed by SDS-PAGE, a band showing a molecular weight of approximately 50 kilo Daltons under reducing conditions was observed. This band reacted with rabbit anti-MITF antibodies. Note that this rabbit anti-MITF antibody was obtained by affinity purification of an antiserum obtained by immunizing a rabbit with a peptide of the 20 C-terminal amino acid residues of MITF, using a column to which the same peptide was bound, to prepare antibodies that specifically recognize the same peptide.

Formulation Example 1

[0070] A composition comprising the fusion protein of the present invention and Dulbecco's PBS containing 10% glycerol was prepared.

Experimental Example 1

[0071] 1) First, whether or not fusion proteins of the present invention translocate into the cells was comfirmed. A fusion protein of the MITF variant prepared in Example 1 was fluorescence labeled with FITC (fluorescein-isothiocyanate) by conventional methods and added to COS7 cells. One hour after addition, a fluorescence intensity was measured using FACS (fluorescence-activated cell sorter) Calibur (Becton Dickinson). Results are shown in Table 3. TABLE 3 Peak position of fluorescence intensity Added agent (FL1-H) PBS (fusion protein is not 3 added) His Tag-PTD-mi variant of 6 MITF His Tag-PTD-wh variant of 10 MITF

[0072] As is apparent from Table 3, strong fluorescence is observed to be associated with the cells, confirming efficient translocation of the fusion protein into the cells.

[0073] 2) Luciferase analysis was carried out to examine whether the fusion protein of the present invention translocates into cells and inhibits endogenous MITF activity.

[0074] A normal MITF expression plasmid pSU063 (MITF-M/pCDNA3), various vectors for preparing plasmids in which a luciferase gene is connected downstream of the c-kit gene promoter, that is, C-kit-Rluc expression vector pSU053 (C-kit/R-luc), and luciferase expression vector pGL2 (Lluc, Promega) were used as expression plasmids. Each plasmid was introduced (transfected) into COS7 cells. The conditions were as follows. The quantity of plasmid used was 3 μg per 6-cm dish (pSU063: pSU053: pGL2=1:1:0.1). For plasmid introduction, a transfection kit (Stratagene, #200385) was used.

[0075] A quantity of 1×10⁵ cells per well were inoculated in a 6-well plate and left at rest for 17 hours in a CO₂ incubator. After adding 90 μL of distilled water and 3 μg of DNA to 10 μL of Solution 1 (a reagent included in the kit, comprising 2.5 M CaCl₂), an equal quantity of Solution 2 [a reagent included in the kit, comprising 2×PBS (pH 6.95)] was added and this was left stationary at room temperature for 20 minutes. The mixed DNA solution was added to the culture solution. After 24 hours, the culture medium was replaced, and 1 μg/mL of the fusion protein of the present invention (prepared in Example 1) was added.

[0076] Forty-eight hours after addition of the fusion protein of the present invention, cells were washed with PBS (−), 0.5 mL of passive lysis buffer were added per 6-well dish, and cells were collected by scraping, using a scraper. Cells were transferred to a 1.5-mL capacity tube and centrifuged at 14000 rpm at 4° C. for 5 minutes. Measurement of luciferase activity was carried out using a Dual luciferase assay kit (Promega). On a 96-well plate, 100 μL of Luciferase Assay Reagent II was added to and mixed with 20 μL of centrifugation supernatant, and firefly luciferase activity was measured. Furthermore, 100 μL of Stop & Go Reagent was further added to and mixed with the mixture, and a Renilla luciferase (R-luc) activity was measured. R-luc counts were corrected with pGL2 and then converted to a value where the R-luc on PBS addition was taken to be 1. Results are shown in Table 4. TABLE 4 Added agent Luciferase activity pSU053 + pSU063 + PBS taken as 1 pSU053 + pSU063 + mi 0.92 pSU053 + pSU063 + wh 0.72

[0077] Note that, in the table, mi shows the fusion protein comprising the His Tag-PTD-MITF mi variant, and wh shows the fusion protein comprising the His Tag-PTD-MITF wh variant, respectively. The luciferase activity expressed by the COS cells was also inhibited in the presence of the fusion protein of the present invention, suggesting that the fusion protein transfers into the cells and inhibits endogenous MITF.

Experimental Example 2

[0078] Investigation was made into how the fusion protein of the present invention influences an SCF induced mast cell differentiation system.

[0079] Bone marrow cells were prepared from normal mice; of these, 2×10⁶/well were cultured in RPMI 1640 containing 10% FCS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol, 2 μg/mL fusion protein of the present invention (His Tag-PTD-MITF wh variant), in the presence of 50-100 ng/mL bone marrow cell factor (SCF, IBL), in a 24-well plate. Once a week, half the quantity of the culture medium was exchanged, and in so doing, SCF was also added. After culturing at 37° C. for 21 days, a count of mast cells and a representation of c-kit and IgE receptors were measured.

[0080] The count of mast cells was measured by a toluidine blue staining method. The toluidine blue staining was carried out according to Current Protocols in Immunology (Wiley) section 7.25.2, using a staining solution at pH 2.7.

[0081] The representation of c-kit was measured using FACS. Cells were suspended in 100 μL PBS containing 0.1% NaN₃ and 0.1% BSA, and incubated for 1 hour on ice with 5-10 μg/mL of R-PE (R-phycoerythrin)-labeled antimouse c-kit antibody (PharMingen) or R-PE-labeled rat IgG 2B, k isotype standard (PharMingen). After washing, 1 μg/mL of pyridium iodide was added, and analysis was performed with FACS Calibur (above-described). The representation of IgE receptors was measured in the same way using 5 μg/mL FITC-labeled mouse IgE and analyzed with FITC. Results for counts of mast cells are shown in Table 5. TABLE 5 Count of mast cells Fusion protein of the 14th day of 21th day of present invention culture culture no addition 9 × 10⁴ 3 × 10⁶ addition 3 × 10⁴ 2 × 10⁵

[0082] In the group to which the fusion protein of the present invention was added, at 21th day of culture, the count of mast cells decreased to no more than 1/10, as compared to the group to which this was not added. Furthermore, results for c-kit expression at 21th day of culture are as shown in Table 6. TABLE 6 Fusion protein of the Peak position of present invention fluorescence intensity no addition 1 × 10³ addition 7 × 10¹

[0083] In the group to which the fusion protein of the present invention was added, at 21th day of culture, the representation of c-kit decreased to no more than 1/10, as compared to the group to which this was not added. Furthermore, the representation of IgE receptors did not change (experimental data not shown).

[0084] Thus, it was confirmed that the fusion protein of the present invention specifically inhibits the expression of the SCF receptor c-kit in mast cell precursor cells and, as a result, strongly inhibits the differentiation of mast cells.

Experimental Example 3

[0085] The influence on mast cell survival in co-culture systems with fibroblasts was examined in order to confirm the influence of the fusion protein of the present invention on mature mast cells.

[0086] Splenic cells were prepared from normal mice, cultured in RPMI 1640 containing 10% FCS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol, in the presence of IL-3 (GENZYME) or WEHI-3 cell conditioned culture medium (WEHI-3CM), for no less than 3 weeks, to obtain spleen-derived cultured mast cells (SMC). In a 6-well plate, NIH-3T3 (Riken Cell Bank) fibroblasts were cultured until confluency; the SMC were suspended in the above-mentioned culture medium, not containing IL-3 and WEHI-3CM, but containing 1 μg/mL of the fusion protein of the present invention, and inoculated at 3×10⁵. Every 2 to 3 days, the culture medium was replaced with fresh media. After culturing at 37° C. for 15 days, the mast cell count, histamine quantity, and chymase activity were measured. The various experimental systems are shown in Table 7. TABLE 7 Experimental system Fibroblasts Mast cell Fusion protein A present Normal mast No addition cells B present Same as His Tag-PTD-MITF above mi variant C present Same as His Tag-PTD-MITF above wh variant D present mi mast cells No addition (mi/mi SMC)

[0087] Histamine was quantified by an RIA method (Eiken Chemical). Measurement of the chymase activity inside granules was performed using a specific synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-pNA (Sigma). The results are shown in Table 8. TABLE 8 Count of mast cells Chymase activity Histamine (counts/well) (mOD/minute/well) (ng/well) A 3 × 10⁴ 14 1.05 B 1 × 10⁴ 4 0.25 C 7 × 10³ 3 0.2 D 8 × 10² 0 0.03

[0088] When fibroblasts and normal mast cells were co-cultured, the mast cells survived for a long period of time. Furthermore, mi mast cells which were artificially constructed, having an anomaly in MITF(mi/mi SMC, which do not exist naturally) died within 2 weeks due to apoptosis in a co-culture with fibroblasts. When the fusion protein of the present invention (mi variant type and wh variant type) was added to a co-culture of normal mast cells and fibroblasts, survival of mast cells was inhibited, and most died after 2 weeks in the same manner as with the mi mast cells. A low count of mast cells was also confirmed by the decrease in the quantity of histamine and chymase activity. The result suggests that the fusion protein of the present invention induces apoptosis in mature mast cells.

Experimental Example 4

[0089] The influence of the concentration at which the fusion protein of the present invention is added was examined. An MC/9 mast cell strain (obtained from ATCC) was incubated on ice for 1 hour in an FACS staining buffer solution (Dulbecco's PBS containing 0.1% bovine serum albumin and 0.1% sodium azide) in the presence or the absence of a fusion protein comprising an FITC-labeled His Tag-PTD-MITF wh variant. After washing, pyridium iodide was added to gate out the dead cells, and this was analyzed with the FACS Calibur (above-described). Results are shown in Table 9. TABLE 9 Concentration of added Peak position of fusion protein fluorescence intensity   0 (μg/mL) 2.5  2.5 3.5 12.6 7   63 22

[0090] From results of Table 9, it is understood that, depending on the concentration at which the fusion protein of the present invention is added, the fusion protein translocated into cells at higher concentrations.

Example 2 His Tag-PTD-HLH

[0091] 1) Construction of the Expression Vector

[0092] A normal M-type MITF (pSU054) was used as the template for the construction of the expression vector. For the HLH fragment, a region corresponding to N-terminal residues 196 to 285 of the M-type was amplified by the PCR method. His Tag, PTD, and the recognition sequence for the restriction endonuclease NcoI were added on the N-terminal side of the amplified fragment, using primers (Tat1, Tat2, Tat3, and HLH F). Furthermore, the recognition sequence for the restriction endonuclease XbaI was added on the C-terminal side. The amplified fragment was digested with NcoI+XbaI and replaced with the NcoI-XbaI region of pSU081 constructed in Example 1. The amplified fragment was TA-cloned, and it was verified that the base sequence was correct. The constructed vector, pSU085, comprises the lac promoter, the DNA for a His Tag-PTD-HLH region fusion protein, and amp^(r) (FIG. 2).

[0093] A primer HLHF has the following base sequence. HLHF (SEQ ID NO: 25): GCGACGAAGAGGTATGTTGGCTAAAGAGA GG

[0094] 2) The fusion protein was produced and purified as in Example 1.

Example 3 His Tag-PTD-A-type N-terminal Region of MITF

[0095] 1) Construction of the Expression Vector

[0096] PCR was carried out using two types of primers (pTD3-MITF a and MITF R-N), with pSU064 (A-type MITF/pCDNA3, see WO 01/66735) as the template to amplify the N-terminal region (N-terminal amino acids 1 to 305). A SacII site at the 5′ end of the pTD3-MITFa and an EcoRI site at the 5′ end of MITF R-N were added. The amplified fragment was digested by SacII+EcoRI and inserted between the same sites on pSU093 (PTD cassette expression vector with pTrcHis as the basic scaffold) (FIG. 3). In the constructed expression vector pSU087, DNA fragments are connected in the His Tag-PTD-A-type N-terminal region (of MITF), and the fusion protein is expressed under the regulation of the lac promoter.

[0097] Primers had following base sequences. pTD3-MITF a (SEQ ID NO: 26): CGCCGCGGAATGCAGTCCGAATCGGGAATC MITF R-N (SEQ ID NO: 27): GAATTCACTATGCTCTTGCTTCAGACTCTGTGG GG

[0098] 2) After producing the fusion protein according to Example 1, purification thereof was performed using a TALON (product name; CLONTECH) instead of the Ni-agarose. The purified product was prepared as a 10% glycerol/PBS solution.

Example 4 Construction of an Expression Vector Using pET14b

[0099] pSU083 (wh form) NcoI-Noel fragment and same region in pET14b (made by linking T7 promoter, His Tag, and amp^(r); Takara) were exchanged to construct the expression vector pSU121 (FIG. 4). This pSU121 has the His Tag, the PTD sequence, and the wh type MITF connected downstream of a T7 promoter and expresses the fusion protein (His Tag-PTD-wh type MITF) under the regulation of the T7 promoter inside Escherichia coli cells.

[0100]Escherichia coli was cultured in a flask to produce the fusion protein (wh form), according to Example 1. This was purified using Ni-NTA agarose according to Example 1, whereafter urea removal processing was carried out using a Slide-A-Dialyzer (Pierce). A 290 mM sorbitol, 10 μM EDTA, 1 mM tris-hydroxymethyl-aminomethane buffer solution (pH 8) was used as a solvent. Approximately 10 mg of fusion protein was purified per liter of culture solution.

Example 5 Purification Process (HLH)

[0101] The fusion protein (HLH form) was eluted from a Q-Sepharose column with 1 M NaCl/20 mM HEPES (pH 8), and recovered fractions were buffer-exchanged with PBS using a PD-10 column (Pharmacia) prior to use.

Example 6 High-Density Culture (wh form)

[0102] The expression vector pSU083 was introduced into HB101 strain Escherichia coli to prepare transformants. This was cultured using EBM0010010 culture medium (ampicillin added) in a jar fermenter at 37° C. for 16 hours to produce the fusion protein. Purification was performed according to Example 1.

Example 7 High-Density Culture (HLH)

[0103] The fusion protein was produced using pSU085 according to Example 6. In carrying out purification according to Example 1, the following points were modified: 6 M guanidine hydrochloride was used instead of 8 M urea during Ni-NTA agarose treatment, and instead of the anion exchange treatment, a 3000 molecular weight cutoff membrane was used to concentrate the protein; then, this was desalted over a PD-10 column (Pharmacia) equilibrated with 0.5 M NaCl/20 mM HEPES (pH 8). This was dialyzed against 0.5 M NaCl/20 mM HEPES (pH 7.4), aliquated, and stored at −80° C. For in vivo use, this was diluted to obtain NaCl concentration of 0.15 M and then used.

Formulation Example 2

[0104] A composition was prepared, comprising the fusion protein, 10% glycerol, and PBS.

Formulation Example 3

[0105] This was prepared as a solution of fusion protein, 0.15-1 M NaCl, and 20 mM HEPES (pH 7.4-8).

Experimental Example 5 (Purification Result)

[0106] Various fusion proteins were purified from Escherichia coli cells; molecular weights and yields thereof (per 4-L flask culture) are shown in Table 10. TABLE 10 Molecular Fusion protein weight (kDa) Yields (μg) mi form 50 630 wh form 50 400 HLH form 14 130

[0107] The molecular weight of the fusion protein (A-type N-terminal region) was 37 kDa.

Experimental Example 6 (in Vitro Inhibition)

[0108] Experiments were carried out according to Experimental Example 3. After the action of 10 nM fusion protein for 17 days, the mast cell count, histamine, tryptase, and chymase were measured. Results are shown in Table 11. TABLE 11 Vehicle (solvent HLH series alone) Count of mast cells (cells/well)   6 ± 1**(×10⁴)   25 ± 1 (×10⁴) histamine (ng/well)   52 ± 4**   90 ± 4 tryptase (mOD/minute)  2.3 ± 0.4** 10.6 ± 1.2 chymase (mOD/minute) 0.23 ± 0.08** 0.78 ± 0.10

Experimental Example 7 (Same as Above)

[0109] Experiments were carried out according to Experimental Example 3. After the action of 20 nM fusion protein for 15 days, a ratio of mast cells was measured. Results are shown in Table 12. The fusion protein of the present invention suppressed survival of mast cells. TABLE 12 Fusion protein Ratio of mast cells (%) mi form 0.9 ± 0.1** wh form 0.7 ± 0.2** HLH form 0.8 ± 0.1** Vehicle 1.4 ± 0.1**

Experimental Example 8 (Same as Above)

[0110] 1) Experiments were carried out according to Experimental Example 2. The count of mast cells was determined when the fusion protein was added to mouse bone marrow cells (2×10⁶/well) in the presence of SCF and cultured for 28 days. The number of wells was 3. Results are shown in Table 13. TABLE 13 Fusion protein Addition concentration count of mast cells vehicle (PBS) 8 × 10⁶ wh form 40 (nM) 3 × 10⁵ HLH form 70 3 × 10⁵

[0111] 2) Experiments were carried out according to 1). The concentration of fusion protein added was 2.5 nM. Histamine concentration and chymase activity were determined. Results are shown in Table 14. TABLE 14 Histamine HLH form  280 ± 40** (ng/mL) vehicle  740 ± 40 chymase mi type  0.9 ± 0.2* (mOD/minute) HLH type  0.6 ± 0.2** vehicle  1.8 ± 0.2

[0112] 3) Experiments were carried out according to 1). The concentration of fusion protein added was 2.5-20 nM. Cell counts and chymase activity were determined. The number of experiments was 2. Results are shown in Table 15. TABLE 15 Cell count Chymase (mOD/minute) Fusion A-type N- A-type N- protein terminus HLH terminus HLH vehicle  10 × 10⁶  10 × 10⁶ 10 10 2.5 (nM) 0.3 × 10⁶   5 × 10⁶ 0.2 3   5 0.2 × 10⁶   6 × 10⁶ 0.05 1  10 0.4 × 10⁶   1 × 10⁶ 0.06 0.09  20 0.4 × 10⁶ 0.6 × 10⁶ 0.03 0.03

Experimental Example 9 (Effect on Human Mast Cells)

[0113] 1) A density of 2×10⁴/well human CD34-positive bone marrow cells (BioWhittaker) were cultured for 9 weeks, replacing half the quantity of a culture solution to which SCF (100 ng/ml), IL-6 (100 ng/ml), IL-10 (10 ng/ml), and fusion protein (HLH type) were added once a week; and cell counts, as well as chymase activity, and quantity of histamine in the collected cell lysate were measured. The culture solution used was Media I containing 5% FCS (IBL Co., Ltd.). The number of experiments was 3. Results were that the fusion protein of the present invention inhibited human mast cell differentiation (Table 16). TABLE 16 Fusion protein Vehicle cell count (at maximum)   6 × 10⁵   4 × 10⁵ chymase (mOD/minute) 0.07 ± 0.02 0.25 ± 0.07 histamine (ng/mL)   40 ± 20   70 ± 15

[0114] 2) Human CD34-positive bone marrow cells were cultured for 8 weeks according to 1) in the presence of cytokines to prepare partially differentiated immature mast cells. Cells were temporarily recovered, washed by centrifugation, and used to inoculate a culture solution on a new plate at 7×10⁵well; culturing was continued, using the same culture solution (containing the above-mentioned cytokines) to which fusion protein was added (10 or 50 nM of HLH form were added).

[0115] Once a week, half the quantity of the culture solution was replaced. After 2 weeks of culture, the cell count, chymase activity, and quantity of histamine were measured. Furthermore, the cells were stained with PE-labeled anti c-kit antibody (or PE-labeled control antibody) and analyzed by FACS. The results were that the fusion protein of the present invention had an effect on immature mast cells, inhibiting c-kit expression and inhibiting the maturation of human mast cells (Table 17). TABLE 17 Fusion Immediately protein Same before culture 50 nM 10 nM Vehicle chymase 0.08 0.03 0.06 0.15 (mOD/minute) histamine (ng/mL) 290 200 330 570 peak position of 8 × 10 1 × 10² 2 × 10² FACS

[0116] 3) Human CD34-positive bone marrow cells were cultured according to 1) in the presence of cytokines for 12 weeks to prepare completely differentiated mature mast cells. Cells were temporarily collected, washed by centrifugation, and used to inoculate in a culture solution on a new plate at 2.4×10⁴/well; culture was continued, using the same culture solution (containing the above-mentioned cytokines) to which fusion protein was added (2, 10, or 50 nM of HLH form were added). Once a week, half the quantity of the culture solution was replaced. After 3 weeks of culture, the cell count and the quantity of histamine were measured. Results were that the fusion protein of the present invention decreased the quantity of histamine in a concentration-dependent manner, suppressing the function of the cultured mature mast cells from human bone marrow (Table 18). TABLE 18 Fusion protein (nM) Histamine (ng/mL) solvent only 600 ± 20  2 520 ± 50 10 370 ± 10 50 300 ± 10

Experimental Example 10 (in Vivo Inhibition)

[0117] 1) A dose of 60 μg fusion protein (wh form) in 200 μL PBS was administered into peritoneal cavities of C57BU6 mice 3 times a week for 4 weeks. The day after final administration, peritoneal cells were collected, and following items were measured. The number of mice/group was 4. Results from the 4-week administration are shown in Table 19.

[0118] Total cell count: This was measured using coulter counter or hemocytometric counter.

[0119] Mast cells: A smear specimen was stained with toluidine blue, and the proportion of positives was calculated.

[0120] Histamine: Triton X-100 was added to a cell suspension of peritoneal cavity to prepare a lysate, and the lysate was measured with an ELISA kit.

[0121] Cells positive for both c-kit and IgE receptors: Collected peritoneal cavity cells were double-stained with PE-labeled anti c-kit antibody and mouse IgE/biotin-labeled anti-IgE antibody/APC-labeled streptavidin and analyzed by FACS. TABLE 19 Vehicle Fusion Significant group protein difference Ratio of mast cells (%) 0.8 0.4 0.0394 Count of mast cells 0.3 × 10⁵ 0.17 × 10⁵ 0.2784 Frequency of c-kit⁺ 0.6 0.4 0.3080 IgER⁺ cells (%) Count of same cells 2.3 × 10⁵  1.4 × 10⁵ 0.2214 histamine (ng/10⁶ cells) 42 11 0.0510

[0122] 2) A quantity of 10 or 50 μg of fusion protein (wh form, both in 200 μL of vehicle) was administered into peritoneal cavities of mice for 2 weeks. The number of mice/group was 10. Each item was measured as in 1). Results are shown in Table 20. TABLE 20 Fusion Fusion Vehicle protein protein group 10 μg 50 μg Count of total cells   9 ± 1(×10⁶)   11 ± 1(×10⁶)   12 ± 1*(×10⁶) Ratio of mast cells  0.9 ± 0.2  0.4 ± 0.1**  0.2 ± 0.1** (%) Ratio of both- 0.48 ± 0.10 0.29 ± 0.05 0.20 ± 0.04** positive mast cells (%) Fluorescence  900 ± 50  680 ± 30**  670 ± 30** intensity of c-kit average

[0123] 3) A quantity of 10 μg of fusion protein (HLH form) in 350 μL of vehicle was administered into peritoneal cavities of mice for 13 days. The mast cell count and the quantity of histamine were measured according to 1). Results are shown in Table 21. TABLE 21 Vehicle group Fusion protein mast cell count 1.9 ± 0.6(×10⁴) 0.1 ± 0.1**(×10⁴) histamine (nM) 6.7 ± 2.0 0.7 ± 0.5*

[0124] The fusion protein of the present invention (wh form, HLH form) decreased the in vivo mast cell count in both experimental systems.

Industrial Applicability

[0125] According to the present invention, cell death of mast cells can be induced using MITF variants.

[0126] Therefore, it is possible to provide the clinical field with an agent useful in the prevention and treatment of various diseases in which mast cells are implicated.

[0127] Note that the present application claims priority from Japanese Patent Application No. 2001-204567.

1 29 1 415 PRT Mus musculus misc_feature MITF mi mutant 1 Met Leu Glu Tyr Ser His Tyr Gln Val Gln Thr His Leu Glu Asn Pro 1 5 10 15 Thr Lys Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr 20 25 30 Leu Ser Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser 35 40 45 Pro Cys Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly 50 55 60 Ser Ser Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn 65 70 75 80 Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu 85 90 95 Ser Glu Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln 100 105 110 Met Asp Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn 115 120 125 Glu Glu Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr 130 135 140 Leu Pro Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu 145 150 155 160 Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro 165 170 175 Asn Ile Lys Arg Glu Leu Thr Ala Cys Ile Phe Pro Thr Glu Ser Glu 180 185 190 Ala Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu 195 200 205 Ile Glu Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly 210 215 220 Thr Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly 225 230 235 240 Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu 245 250 255 Gln Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His 260 265 270 Ala Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu Glu Met Gln Ala 275 280 285 Arg Ala His Gly Leu Ser Leu Ile Pro Ser Thr Gly Leu Cys Ser Pro 290 295 300 Asp Leu Val Asn Arg Ile Ile Lys Gln Glu Pro Val Leu Glu Asn Cys 305 310 315 320 Ser Gln Glu Leu Val Gln His Gln Ala Asp Leu Thr Cys Thr Thr Thr 325 330 335 Leu Asp Leu Thr Asp Gly Thr Ile Thr Phe Thr Asn Asn Leu Gly Thr 340 345 350 Met Pro Glu Ser Ser Pro Ala Tyr Ser Ile Pro Arg Lys Met Gly Ser 355 360 365 Asn Leu Glu Asp Ile Leu Met Asp Asp Ala Leu Ser Pro Val Gly Val 370 375 380 Thr Asp Pro Leu Leu Ser Ser Val Ser Pro Gly Ala Ser Lys Thr Ser 385 390 395 400 Ser Arg Arg Ser Ser Met Ser Ala Glu Glu Thr Glu His Ala Cys 405 410 415 2 1248 DNA Mus musculus misc_feature DNA coding MITF mi mutant 2 atg cta gaa tac agt cac tac cag gtg cag acc cac ctg gaa aac ccc 48 Met Leu Glu Tyr Ser His Tyr Gln Val Gln Thr His Leu Glu Asn Pro 1 5 10 15 acc aag tac cac ata cag caa gct cag agg cac cag gta aag cag tac 96 Thr Lys Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr 20 25 30 ctt tct acc act tta gca aat aaa cat gcc agc caa gtc ctg agc tca 144 Leu Ser Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser 35 40 45 cca tgt cca aac cag cct ggc gac cat gcc atg cca cca gtg ccg ggg 192 Pro Cys Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly 50 55 60 agc agc gca ccc aac agc cct atg gct atg ctc act ctt aac tcc aac 240 Ser Ser Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn 65 70 75 80 tgt gaa aaa gag gca ttt tat aag ttt gag gag cag agc agg gca gag 288 Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu 85 90 95 agt gag tgc cca ggt atg aac acg cac tct cga gcg tcg tgc atg cag 336 Ser Glu Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln 100 105 110 atg gat gat gta att gat gac atc atc agc ctg gaa tca agt tat aat 384 Met Asp Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn 115 120 125 gaa gaa att ttg ggc ttg atg gat ccg gcc ttg caa atg gca aat acg 432 Glu Glu Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr 130 135 140 tta ccc gtc tct gga aac ttg atc gac ctc tac agc aac cag ggc ctg 480 Leu Pro Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu 145 150 155 160 cca ccg cca ggc ctt acc atc agc aac tcc tgt cca gcc aac ctt ccc 528 Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro 165 170 175 aac ata aaa agg gag ctc aca gcg tgt att ttc ccc aca gag tct gaa 576 Asn Ile Lys Arg Glu Leu Thr Ala Cys Ile Phe Pro Thr Glu Ser Glu 180 185 190 gca aga gca ttg gct aaa gag agg cag aaa aag gac aat cac aac ttg 624 Ala Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu 195 200 205 att gaa cga aga aga ttt aac ata aac gac cgc att aag gag cta ggt 672 Ile Glu Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly 210 215 220 act ctg atc ccc aag tca aat gat cca gac atg cgg tgg aac aag gga 720 Thr Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly 225 230 235 240 acc att ctc aag gcc tct gtg gac tac atc cgg aag ttg caa cgg gaa 768 Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu 245 250 255 cag caa cga gct aag gac ctt gaa aac cga cag aag aag ctg gag cat 816 Gln Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His 260 265 270 gcg aac cgg cac ctg ctg ctc aga gta cag gag ctg gag atg cag gct 864 Ala Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu Glu Met Gln Ala 275 280 285 aga gcg cat gga ctt tcc ctt atc cca tcc acc ggt ctc tgc tcg cct 912 Arg Ala His Gly Leu Ser Leu Ile Pro Ser Thr Gly Leu Cys Ser Pro 290 295 300 gat ctg gtg aat cgg atc atc aag caa gaa cca gtt ctt gag aac tgc 960 Asp Leu Val Asn Arg Ile Ile Lys Gln Glu Pro Val Leu Glu Asn Cys 305 310 315 320 agc cag gaa ctt gta cag cac cag gca gac ctg aca tgt acg aca act 1008 Ser Gln Glu Leu Val Gln His Gln Ala Asp Leu Thr Cys Thr Thr Thr 325 330 335 ctg gat ctc acg gac ggt acc atc acc ttt acc aac aac ctc ggc acc 1056 Leu Asp Leu Thr Asp Gly Thr Ile Thr Phe Thr Asn Asn Leu Gly Thr 340 345 350 atg ccg gag agc agc ccg gcc tac agc atc ccc agg aag atg ggc tcc 1104 Met Pro Glu Ser Ser Pro Ala Tyr Ser Ile Pro Arg Lys Met Gly Ser 355 360 365 aac ttg gaa gac atc ctg atg gac gat gcc ctc tca cct gtt gga gtc 1152 Asn Leu Glu Asp Ile Leu Met Asp Asp Ala Leu Ser Pro Val Gly Val 370 375 380 acc gac cca ctg ctg tca tca gtg tcg cca gga gct tca aaa aca agc 1200 Thr Asp Pro Leu Leu Ser Ser Val Ser Pro Gly Ala Ser Lys Thr Ser 385 390 395 400 agc cgg agg agc agt atg agc gca gaa gaa acg gag cat gcg tgt tag 1248 Ser Arg Arg Ser Ser Met Ser Ala Glu Glu Thr Glu His Ala Cys 405 410 415 3 410 PRT Mus musculus misc_feature MITF wh mutant 3 Met Leu Glu Tyr Ser His Tyr Gln Val Gln Thr His Leu Glu Asn Pro 1 5 10 15 Thr Lys Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr 20 25 30 Leu Ser Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser 35 40 45 Pro Cys Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly 50 55 60 Ser Ser Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn 65 70 75 80 Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu 85 90 95 Ser Glu Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln 100 105 110 Met Asp Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn 115 120 125 Glu Glu Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr 130 135 140 Leu Pro Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu 145 150 155 160 Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro 165 170 175 Asn Ile Lys Arg Glu Leu Thr Glu Ser Glu Ala Arg Ala Leu Ala Lys 180 185 190 Glu Arg Gln Lys Lys Asp Asn His Asn Leu Asn Glu Arg Arg Arg Arg 195 200 205 Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys 210 215 220 Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala 225 230 235 240 Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys 245 250 255 Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu 260 265 270 Leu Leu Arg Val Gln Glu Leu Glu Met Gln Ala Arg Ala His Gly Leu 275 280 285 Ser Leu Ile Pro Ser Thr Gly Leu Cys Ser Pro Asp Leu Val Asn Arg 290 295 300 Ile Ile Lys Gln Glu Pro Val Leu Glu Asn Cys Ser Gln Glu Leu Val 305 310 315 320 Gln His Gln Ala Asp Leu Thr Cys Thr Thr Thr Leu Asp Leu Thr Asp 325 330 335 Gly Thr Ile Thr Phe Thr Asn Asn Leu Gly Thr Met Pro Glu Ser Ser 340 345 350 Pro Ala Tyr Ser Ile Pro Arg Lys Met Gly Ser Asn Leu Glu Asp Ile 355 360 365 Leu Met Asp Asp Ala Leu Ser Pro Val Gly Val Thr Asp Pro Leu Leu 370 375 380 Ser Ser Val Ser Pro Gly Ala Ser Lys Thr Ser Ser Arg Arg Ser Ser 385 390 395 400 Met Ser Ala Glu Glu Thr Glu His Ala Cys 405 410 4 1233 DNA Mus musculus misc_feature DNA coding MITF wh mutant 4 atg cta gaa tac agt cac tac cag gtg cag acc cac ctg gaa aac ccc 48 Met Leu Glu Tyr Ser His Tyr Gln Val Gln Thr His Leu Glu Asn Pro 1 5 10 15 acc aag tac cac ata cag caa gct cag agg cac cag gta aag cag tac 96 Thr Lys Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr 20 25 30 ctt tct acc act tta gca aat aaa cat gcc agc caa gtc ctg agc tca 144 Leu Ser Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser 35 40 45 cca tgt cca aac cag cct ggc gac cat gcc atg cca cca gtg ccg ggg 192 Pro Cys Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly 50 55 60 agc agc gca ccc aac agc cct atg gct atg ctc act ctt aac tcc aac 240 Ser Ser Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn 65 70 75 80 tgt gaa aaa gag gca ttt tat aag ttt gag gag cag agc agg gca gag 288 Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu 85 90 95 agt gag tgc cca ggt atg aac acg cac tct cga gcg tcg tgc atg cag 336 Ser Glu Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln 100 105 110 atg gat gat gta att gat gac atc atc agc ctg gaa tca agt tat aat 384 Met Asp Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn 115 120 125 gaa gaa att ttg ggc ttg atg gat ccg gcc ttg caa atg gca aat acg 432 Glu Glu Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr 130 135 140 tta ccc gtc tct gga aac ttg atc gac ctc tac agc aac cag ggc ctg 480 Leu Pro Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu 145 150 155 160 cca ccg cca ggc ctt acc atc agc aac tcc tgt cca gcc aac ctt ccc 528 Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro 165 170 175 aac ata aaa agg gag ctc aca gag tct gaa gca aga gca ttg gct aaa 576 Asn Ile Lys Arg Glu Leu Thr Glu Ser Glu Ala Arg Ala Leu Ala Lys 180 185 190 gag agg cag aaa aag gac aat cac aac ttg aat gaa cga aga aga aga 624 Glu Arg Gln Lys Lys Asp Asn His Asn Leu Asn Glu Arg Arg Arg Arg 195 200 205 ttt aac ata aac gac cgc att aag gag cta ggt act ctg atc ccc aag 672 Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys 210 215 220 tca aat gat cca gac atg cgg tgg aac aag gga acc att ctc aag gcc 720 Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala 225 230 235 240 tct gtg gac tac atc cgg aag ttg caa cgg gaa cag caa cga gct aag 768 Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys 245 250 255 gac ctt gaa aac cga cag aag aag ctg gag cat gcg aac cgg cac ctg 816 Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu 260 265 270 ctg ctc aga gta cag gag ctg gag atg cag gct aga gcg cat gga ctt 864 Leu Leu Arg Val Gln Glu Leu Glu Met Gln Ala Arg Ala His Gly Leu 275 280 285 tcc ctt atc cca tcc acc ggt ctc tgc tcg cct gat ctg gtg aat cgg 912 Ser Leu Ile Pro Ser Thr Gly Leu Cys Ser Pro Asp Leu Val Asn Arg 290 295 300 atc atc aag caa gaa cca gtt ctt gag aac tgc agc cag gaa ctt gta 960 Ile Ile Lys Gln Glu Pro Val Leu Glu Asn Cys Ser Gln Glu Leu Val 305 310 315 320 cag cac cag gca gac ctg aca tgt acg aca act ctg gat ctc acg gac 1008 Gln His Gln Ala Asp Leu Thr Cys Thr Thr Thr Leu Asp Leu Thr Asp 325 330 335 ggt acc atc acc ttt acc aac aac ctc ggc acc atg ccg gag agc agc 1056 Gly Thr Ile Thr Phe Thr Asn Asn Leu Gly Thr Met Pro Glu Ser Ser 340 345 350 ccg gcc tac agc atc ccc agg aag atg ggc tcc aac ttg gaa gac atc 1104 Pro Ala Tyr Ser Ile Pro Arg Lys Met Gly Ser Asn Leu Glu Asp Ile 355 360 365 ctg atg gac gat gcc ctc tca cct gtt gga gtc acc gac cca ctg ctg 1152 Leu Met Asp Asp Ala Leu Ser Pro Val Gly Val Thr Asp Pro Leu Leu 370 375 380 tca tca gtg tcg cca gga gct tca aaa aca agc agc cgg agg agc agt 1200 Ser Ser Val Ser Pro Gly Ala Ser Lys Thr Ser Ser Arg Arg Ser Ser 385 390 395 400 atg agc gca gaa gaa acg gag cat gcg tgt tag 1233 Met Ser Ala Glu Glu Thr Glu His Ala Cys 405 410 5 91 PRT Artificial Description of Artificial Sequence MITF bHLH-Zip fragment 5 Met Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu Ile Glu 1 5 10 15 Arg Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Thr 20 25 30 Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly Thr 35 40 45 Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu Gln 50 55 60 Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His Ala 65 70 75 80 Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu 85 90 6 276 DNA Artificial Description of Artificial Sequence DNA coding MITF bHLH-Zip fragment 6 atg ttg gct aaa gag agg cag aaa aag gac aat cac aac ttg att gaa 48 Met Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His Asn Leu Ile Glu 1 5 10 15 cga aga aga aga ttt aac ata aac gac cgc att aag gag cta ggt act 96 Arg Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys Glu Leu Gly Thr 20 25 30 ctg atc ccc aag tca aat gat cca gac atg cgg tgg aac aag gga acc 144 Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp Asn Lys Gly Thr 35 40 45 att ctc aag gcc tct gtg gac tac atc cgg aag ttg caa cgg gaa cag 192 Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu Gln Arg Glu Gln 50 55 60 caa cga gct aag gac ctt gaa aac cga cag aag aag ctg gag cat gcg 240 Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys Leu Glu His Ala 65 70 75 80 aac cgg cac ctg ctg ctc aga gta cag gag ctg tag 276 Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu 85 90 7 11 PRT Artificial Description of Artificial Sequence TAT-derived peptide 7 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 8 33 DNA Artificial Description of Artificial Sequence DNA coding TAT-derived peptide 8 tat ggc agg aag aag cgg aga cag cga cga aga 33 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 9 10 PRT Artificial Description of Artificial Sequence HisTag 9 Met Gly Gly Ser His His His His His His 1 5 10 10 30 DNA Artificial Description of Artificial Sequence DNA coding HisTag 10 atg ggg ggt tct cat cat cat cat cat cat 30 Met Gly Gly Ser His His His His His His 1 5 10 11 439 PRT Artificial Description of Artificial Sequence fused protein composed of HisTag-PTD-MITF mi mutant 11 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Glu Tyr Ser His Tyr Gln 20 25 30 Val Gln Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala 35 40 45 Gln Arg His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys 50 55 60 His Ala Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp 65 70 75 80 His Ala Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met 85 90 95 Ala Met Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys 100 105 110 Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr 115 120 125 His Ser Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile 130 135 140 Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp 145 150 155 160 Pro Ala Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile 165 170 175 Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser 180 185 190 Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Ala 195 200 205 Cys Ile Phe Pro Thr Glu Ser Glu Ala Arg Ala Leu Ala Lys Glu Arg 210 215 220 Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Phe Asn Ile 225 230 235 240 Asn Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp 245 250 255 Pro Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp 260 265 270 Tyr Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu 275 280 285 Asn Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu Leu Leu Arg 290 295 300 Val Gln Glu Leu Glu Met Gln Ala Arg Ala His Gly Leu Ser Leu Ile 305 310 315 320 Pro Ser Thr Gly Leu Cys Ser Pro Asp Leu Val Asn Arg Ile Ile Lys 325 330 335 Gln Glu Pro Val Leu Glu Asn Cys Ser Gln Glu Leu Val Gln His Gln 340 345 350 Ala Asp Leu Thr Cys Thr Thr Thr Leu Asp Leu Thr Asp Gly Thr Ile 355 360 365 Thr Phe Thr Asn Asn Leu Gly Thr Met Pro Glu Ser Ser Pro Ala Tyr 370 375 380 Ser Ile Pro Arg Lys Met Gly Ser Asn Leu Glu Asp Ile Leu Met Asp 385 390 395 400 Asp Ala Leu Ser Pro Val Gly Val Thr Asp Pro Leu Leu Ser Ser Val 405 410 415 Ser Pro Gly Ala Ser Lys Thr Ser Ser Arg Arg Ser Ser Met Ser Ala 420 425 430 Glu Glu Thr Glu His Ala Cys 435 12 1320 DNA Artificial Description of Artificial Sequence DNA coding the fused protein composed of HisTag-PTD-MITF mi mutant 12 atg ggg ggt tct cat cat cat cat cat cat ggt ggt tat ggc agg aag 48 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 aag cgg aga cag cga cga aga ggt atg cta gaa tac agt cac tac cag 96 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Glu Tyr Ser His Tyr Gln 20 25 30 gtg cag acc cac ctg gaa aac ccc acc aag tac cac ata cag caa gct 144 Val Gln Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala 35 40 45 cag agg cac cag gta aag cag tac ctt tct acc act tta gca aat aaa 192 Gln Arg His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys 50 55 60 cat gcc agc caa gtc ctg agc tca cca tgt cca aac cag cct ggc gac 240 His Ala Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp 65 70 75 80 cat gcc atg cca cca gtg ccg ggg agc agc gca ccc aac agc cct atg 288 His Ala Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met 85 90 95 gct atg ctc act ctt aac tcc aac tgt gaa aaa gag gca ttt tat aag 336 Ala Met Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys 100 105 110 ttt gag gag cag agc agg gca gag agt gag tgc cca ggt atg aac acg 384 Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr 115 120 125 cac tct cga gcg tcg tgc atg cag atg gat gat gta att gat gac atc 432 His Ser Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile 130 135 140 atc agc ctg gaa tca agt tat aat gaa gaa att ttg ggc ttg atg gat 480 Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp 145 150 155 160 ccg gcc ttg caa atg gca aat acg tta ccc gtc tct gga aac ttg atc 528 Pro Ala Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile 165 170 175 gac ctc tac agc aac cag ggc ctg cca ccg cca ggc ctt acc atc agc 576 Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser 180 185 190 aac tcc tgt cca gcc aac ctt ccc aac ata aaa agg gag ctc aca gcg 624 Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Ala 195 200 205 tgt att ttc ccc aca gag tct gaa gca aga gca ttg gct aaa gag agg 672 Cys Ile Phe Pro Thr Glu Ser Glu Ala Arg Ala Leu Ala Lys Glu Arg 210 215 220 cag aaa aag gac aat cac aac ttg att gaa cga aga aga ttt aac ata 720 Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Phe Asn Ile 225 230 235 240 aac gac cgc att aag gag cta ggt act ctg atc ccc aag tca aat gat 768 Asn Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp 245 250 255 cca gac atg cgg tgg aac aag gga acc att ctc aag gcc tct gtg gac 816 Pro Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp 260 265 270 tac atc cgg aag ttg caa cgg gaa cag caa cga gct aag gac ctt gaa 864 Tyr Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu 275 280 285 aac cga cag aag aag ctg gag cat gcg aac cgg cac ctg ctg ctc aga 912 Asn Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu Leu Leu Arg 290 295 300 gta cag gag ctg gag atg cag gct aga gcg cat gga ctt tcc ctt atc 960 Val Gln Glu Leu Glu Met Gln Ala Arg Ala His Gly Leu Ser Leu Ile 305 310 315 320 cca tcc acc ggt ctc tgc tcg cct gat ctg gtg aat cgg atc atc aag 1008 Pro Ser Thr Gly Leu Cys Ser Pro Asp Leu Val Asn Arg Ile Ile Lys 325 330 335 caa gaa cca gtt ctt gag aac tgc agc cag gaa ctt gta cag cac cag 1056 Gln Glu Pro Val Leu Glu Asn Cys Ser Gln Glu Leu Val Gln His Gln 340 345 350 gca gac ctg aca tgt acg aca act ctg gat ctc acg gac ggt acc atc 1104 Ala Asp Leu Thr Cys Thr Thr Thr Leu Asp Leu Thr Asp Gly Thr Ile 355 360 365 acc ttt acc aac aac ctc ggc acc atg ccg gag agc agc ccg gcc tac 1152 Thr Phe Thr Asn Asn Leu Gly Thr Met Pro Glu Ser Ser Pro Ala Tyr 370 375 380 agc atc ccc agg aag atg ggc tcc aac ttg gaa gac atc ctg atg gac 1200 Ser Ile Pro Arg Lys Met Gly Ser Asn Leu Glu Asp Ile Leu Met Asp 385 390 395 400 gat gcc ctc tca cct gtt gga gtc acc gac cca ctg ctg tca tca gtg 1248 Asp Ala Leu Ser Pro Val Gly Val Thr Asp Pro Leu Leu Ser Ser Val 405 410 415 tcg cca gga gct tca aaa aca agc agc cgg agg agc agt atg agc gca 1296 Ser Pro Gly Ala Ser Lys Thr Ser Ser Arg Arg Ser Ser Met Ser Ala 420 425 430 gaa gaa acg gag cat gcg tgt tag 1320 Glu Glu Thr Glu His Ala Cys 435 13 434 PRT Artificial Description of Artificial Sequence fused protein composed of HisTag-PTD-MITF wh mutant 13 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Glu Tyr Ser His Tyr Gln 20 25 30 Val Gln Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala 35 40 45 Gln Arg His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys 50 55 60 His Ala Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp 65 70 75 80 His Ala Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met 85 90 95 Ala Met Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys 100 105 110 Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr 115 120 125 His Ser Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile 130 135 140 Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp 145 150 155 160 Pro Ala Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile 165 170 175 Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser 180 185 190 Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Glu 195 200 205 Ser Glu Ala Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His 210 215 220 Asn Leu Asn Glu Arg Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys 225 230 235 240 Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp 245 250 255 Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu 260 265 270 Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys 275 280 285 Leu Glu His Ala Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu Glu 290 295 300 Met Gln Ala Arg Ala His Gly Leu Ser Leu Ile Pro Ser Thr Gly Leu 305 310 315 320 Cys Ser Pro Asp Leu Val Asn Arg Ile Ile Lys Gln Glu Pro Val Leu 325 330 335 Glu Asn Cys Ser Gln Glu Leu Val Gln His Gln Ala Asp Leu Thr Cys 340 345 350 Thr Thr Thr Leu Asp Leu Thr Asp Gly Thr Ile Thr Phe Thr Asn Asn 355 360 365 Leu Gly Thr Met Pro Glu Ser Ser Pro Ala Tyr Ser Ile Pro Arg Lys 370 375 380 Met Gly Ser Asn Leu Glu Asp Ile Leu Met Asp Asp Ala Leu Ser Pro 385 390 395 400 Val Gly Val Thr Asp Pro Leu Leu Ser Ser Val Ser Pro Gly Ala Ser 405 410 415 Lys Thr Ser Ser Arg Arg Ser Ser Met Ser Ala Glu Glu Thr Glu His 420 425 430 Ala Cys 14 1305 DNA Artificial Description of Artificial Sequence DNA coding the fused protein composed of HisTag-PTD-MITF wh mutant 14 atg ggg ggt tct cat cat cat cat cat cat ggt ggt tat ggc agg aag 48 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 aag cgg aga cag cga cga aga ggt atg cta gaa tac agt cac tac cag 96 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Glu Tyr Ser His Tyr Gln 20 25 30 gtg cag acc cac ctg gaa aac ccc acc aag tac cac ata cag caa gct 144 Val Gln Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala 35 40 45 cag agg cac cag gta aag cag tac ctt tct acc act tta gca aat aaa 192 Gln Arg His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys 50 55 60 cat gcc agc caa gtc ctg agc tca cca tgt cca aac cag cct ggc gac 240 His Ala Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp 65 70 75 80 cat gcc atg cca cca gtg ccg ggg agc agc gca ccc aac agc cct atg 288 His Ala Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met 85 90 95 gct atg ctc act ctt aac tcc aac tgt gaa aaa gag gca ttt tat aag 336 Ala Met Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys 100 105 110 ttt gag gag cag agc agg gca gag agt gag tgc cca ggt atg aac acg 384 Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr 115 120 125 cac tct cga gcg tcg tgc atg cag atg gat gat gta att gat gac atc 432 His Ser Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile 130 135 140 atc agc ctg gaa tca agt tat aat gaa gaa att ttg ggc ttg atg gat 480 Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp 145 150 155 160 ccg gcc ttg caa atg gca aat acg tta ccc gtc tct gga aac ttg atc 528 Pro Ala Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile 165 170 175 gac ctc tac agc aac cag ggc ctg cca ccg cca ggc ctt acc atc agc 576 Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser 180 185 190 aac tcc tgt cca gcc aac ctt ccc aac ata aaa agg gag ctc aca gag 624 Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Glu 195 200 205 tct gaa gca aga gca ttg gct aaa gag agg cag aaa aag gac aat cac 672 Ser Glu Ala Arg Ala Leu Ala Lys Glu Arg Gln Lys Lys Asp Asn His 210 215 220 aac ttg aat gaa cga aga aga aga ttt aac ata aac gac cgc att aag 720 Asn Leu Asn Glu Arg Arg Arg Arg Phe Asn Ile Asn Asp Arg Ile Lys 225 230 235 240 gag cta ggt act ctg atc ccc aag tca aat gat cca gac atg cgg tgg 768 Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp Pro Asp Met Arg Trp 245 250 255 aac aag gga acc att ctc aag gcc tct gtg gac tac atc cgg aag ttg 816 Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr Ile Arg Lys Leu 260 265 270 caa cgg gaa cag caa cga gct aag gac ctt gaa aac cga cag aag aag 864 Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu Asn Arg Gln Lys Lys 275 280 285 ctg gag cat gcg aac cgg cac ctg ctg ctc aga gta cag gag ctg gag 912 Leu Glu His Ala Asn Arg His Leu Leu Leu Arg Val Gln Glu Leu Glu 290 295 300 atg cag gct aga gcg cat gga ctt tcc ctt atc cca tcc acc ggt ctc 960 Met Gln Ala Arg Ala His Gly Leu Ser Leu Ile Pro Ser Thr Gly Leu 305 310 315 320 tgc tcg cct gat ctg gtg aat cgg atc atc aag caa gaa cca gtt ctt 1008 Cys Ser Pro Asp Leu Val Asn Arg Ile Ile Lys Gln Glu Pro Val Leu 325 330 335 gag aac tgc agc cag gaa ctt gta cag cac cag gca gac ctg aca tgt 1056 Glu Asn Cys Ser Gln Glu Leu Val Gln His Gln Ala Asp Leu Thr Cys 340 345 350 acg aca act ctg gat ctc acg gac ggt acc atc acc ttt acc aac aac 1104 Thr Thr Thr Leu Asp Leu Thr Asp Gly Thr Ile Thr Phe Thr Asn Asn 355 360 365 ctc ggc acc atg ccg gag agc agc ccg gcc tac agc atc ccc agg aag 1152 Leu Gly Thr Met Pro Glu Ser Ser Pro Ala Tyr Ser Ile Pro Arg Lys 370 375 380 atg ggc tcc aac ttg gaa gac atc ctg atg gac gat gcc ctc tca cct 1200 Met Gly Ser Asn Leu Glu Asp Ile Leu Met Asp Asp Ala Leu Ser Pro 385 390 395 400 gtt gga gtc acc gac cca ctg ctg tca tca gtg tcg cca gga gct tca 1248 Val Gly Val Thr Asp Pro Leu Leu Ser Ser Val Ser Pro Gly Ala Ser 405 410 415 aaa aca agc agc cgg agg agc agt atg agc gca gaa gaa acg gag cat 1296 Lys Thr Ser Ser Arg Arg Ser Ser Met Ser Ala Glu Glu Thr Glu His 420 425 430 gcg tgt tag 1305 Ala Cys 15 115 PRT Artificial Description of Artificial Sequence fused protein composed of HisTag-PTD-MITF bHLH-Zip fragment 15 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Ala Lys Glu Arg Gln Lys 20 25 30 Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Phe Asn Ile Asn 35 40 45 Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp Pro 50 55 60 Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr 65 70 75 80 Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu Asn 85 90 95 Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu Leu Leu Arg Val 100 105 110 Gln Glu Leu 115 16 348 DNA Artificial Description of Artificial Sequence DNA coding the fused protein composed of HisTag-PTD-MITF bHLH-Zip protein 16 atg ggg ggt tct cat cat cat cat cat cat ggt ggt tat ggc agg aag 48 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 aag cgg aga cag cga cga aga ggt atg ttg gct aaa gag agg cag aaa 96 Lys Arg Arg Gln Arg Arg Arg Gly Met Leu Ala Lys Glu Arg Gln Lys 20 25 30 aag gac aat cac aac ttg att gaa cga aga aga aga ttt aac ata aac 144 Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Phe Asn Ile Asn 35 40 45 gac cgc att aag gag cta ggt act ctg atc ccc aag tca aat gat cca 192 Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys Ser Asn Asp Pro 50 55 60 gac atg cgg tgg aac aag gga acc att ctc aag gcc tct gtg gac tac 240 Asp Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val Asp Tyr 65 70 75 80 atc cgg aag ttg caa cgg gaa cag caa cga gct aag gac ctt gaa aac 288 Ile Arg Lys Leu Gln Arg Glu Gln Gln Arg Ala Lys Asp Leu Glu Asn 85 90 95 cga cag aag aag ctg gag cat gcg aac cgg cac ctg ctg ctc aga gta 336 Arg Gln Lys Lys Leu Glu His Ala Asn Arg His Leu Leu Leu Arg Val 100 105 110 cag gag ctg tag 348 Gln Glu Leu 115 17 35 DNA Artificial Description of Artificial Sequence primer M-tat 17 gcgacgaaga ggtatgctag aatacagtca ctacc 35 18 36 DNA Artificial Description of Artificial Sequence primer tat 3 18 ggcaggaaga agcggagaca gcgacgaaga ggtatg 36 19 35 DNA Artificial Description of Artificial Sequence primer tat 2 19 atcatcatca tggtggttat ggcaggaaga agcgg 35 20 40 DNA Artificial Description of Artificial Sequence primer tat 1 20 taaaccatgg ggggttctca tcatcatcat catcatggtg 40 21 305 PRT Artificial Description of Artificial Sequence MITF A-type N-terminal region (1-305) 21 Met Gln Ser Glu Ser Gly Ile Val Ala Asp Phe Glu Val Gly Glu Glu 1 5 10 15 Phe His Glu Glu Pro Lys Thr Tyr Tyr Glu Leu Lys Ser Gln Pro Leu 20 25 30 Lys Ser Ser Ser Ser Ala Glu His Ser Gly Ala Ser Lys Pro Pro Leu 35 40 45 Ser Ser Ser Thr Met Thr Ser Arg Ile Leu Leu Arg Gln Gln Leu Met 50 55 60 Arg Glu Gln Met Gln Glu Gln Glu Arg Arg Glu Gln Gln Gln Lys Leu 65 70 75 80 Gln Ala Ala Gln Phe Met Gln Gln Arg Val Ala Val Ser Gln Thr Pro 85 90 95 Ala Ile Asn Val Ser Val Pro Thr Thr Leu Pro Ser Ala Thr Gln Val 100 105 110 Pro Met Glu Val Leu Lys Val Gln Thr His Leu Glu Asn Pro Thr Lys 115 120 125 Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr Leu Ser 130 135 140 Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser Pro Cys 145 150 155 160 Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly Ser Ser 165 170 175 Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn Cys Glu 180 185 190 Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu 195 200 205 Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln Met Asp 210 215 220 Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu 225 230 235 240 Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr Leu Pro 245 250 255 Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro 260 265 270 Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile 275 280 285 Lys Arg Glu Leu Thr Ala Cys Ile Phe Pro Thr Glu Ser Glu Ala Arg 290 295 300 Ala 305 22 918 DNA Artificial Description of Artificial Sequence DNA coding MITF A-type N-terminal region (1-305) 22 atg cag tcc gaa tcg gga atc gtg gcg gat ttc gaa gtc ggg gag gag 48 Met Gln Ser Glu Ser Gly Ile Val Ala Asp Phe Glu Val Gly Glu Glu 1 5 10 15 ttt cac gaa gaa ccc aaa acc tat tac gaa ctc aaa agt caa cct ctg 96 Phe His Glu Glu Pro Lys Thr Tyr Tyr Glu Leu Lys Ser Gln Pro Leu 20 25 30 aag agc agc agt tct gca gag cat tct ggg gcc tcc aag cct ccg tta 144 Lys Ser Ser Ser Ser Ala Glu His Ser Gly Ala Ser Lys Pro Pro Leu 35 40 45 agc tcc tcc act atg aca tca cgc atc ttg cta cgc cag caa ctc atg 192 Ser Ser Ser Thr Met Thr Ser Arg Ile Leu Leu Arg Gln Gln Leu Met 50 55 60 cgt gag cag atg cag gag cag gag cgc agg gag cag cag cag aag ctg 240 Arg Glu Gln Met Gln Glu Gln Glu Arg Arg Glu Gln Gln Gln Lys Leu 65 70 75 80 cag gca gcc cag ttc atg caa cag aga gtg gcc gtg agt cag aca cca 288 Gln Ala Ala Gln Phe Met Gln Gln Arg Val Ala Val Ser Gln Thr Pro 85 90 95 gcc ata aac gtc agc gtg ccc acc acc ctt ccc tct gcc acc cag gtg 336 Ala Ile Asn Val Ser Val Pro Thr Thr Leu Pro Ser Ala Thr Gln Val 100 105 110 ccg atg gaa gtc ctt aag gtg cag acc cac ctg gaa aac ccc acc aag 384 Pro Met Glu Val Leu Lys Val Gln Thr His Leu Glu Asn Pro Thr Lys 115 120 125 tac cac ata cag caa gct cag agg cac cag gta aag cag tac ctt tct 432 Tyr His Ile Gln Gln Ala Gln Arg His Gln Val Lys Gln Tyr Leu Ser 130 135 140 acc act tta gca aat aaa cat gcc agc caa gtc ctg agc tca cca tgt 480 Thr Thr Leu Ala Asn Lys His Ala Ser Gln Val Leu Ser Ser Pro Cys 145 150 155 160 cca aac cag cct ggc gac cat gcc atg cca cca gtg ccg ggg agc agc 528 Pro Asn Gln Pro Gly Asp His Ala Met Pro Pro Val Pro Gly Ser Ser 165 170 175 gca ccc aac agc cct atg gct atg ctc act ctt aac tcc aac tgt gaa 576 Ala Pro Asn Ser Pro Met Ala Met Leu Thr Leu Asn Ser Asn Cys Glu 180 185 190 aaa gag gca ttt tat aag ttt gag gag cag agc agg gca gag agt gag 624 Lys Glu Ala Phe Tyr Lys Phe Glu Glu Gln Ser Arg Ala Glu Ser Glu 195 200 205 tgc cca ggt atg aac acg cac tct cga gcg tcg tgc atg cag atg gat 672 Cys Pro Gly Met Asn Thr His Ser Arg Ala Ser Cys Met Gln Met Asp 210 215 220 gat gta att gat gac atc atc agc ctg gaa tca agt tat aat gaa gaa 720 Asp Val Ile Asp Asp Ile Ile Ser Leu Glu Ser Ser Tyr Asn Glu Glu 225 230 235 240 att ttg ggc ttg atg gat ccg gcc ttg caa atg gca aat acg tta ccc 768 Ile Leu Gly Leu Met Asp Pro Ala Leu Gln Met Ala Asn Thr Leu Pro 245 250 255 gtc tct gga aac ttg atc gac ctc tac agc aac cag ggc ctg cca ccg 816 Val Ser Gly Asn Leu Ile Asp Leu Tyr Ser Asn Gln Gly Leu Pro Pro 260 265 270 cca ggc ctt acc atc agc aac tcc tgt cca gcc aac ctt ccc aac ata 864 Pro Gly Leu Thr Ile Ser Asn Ser Cys Pro Ala Asn Leu Pro Asn Ile 275 280 285 aaa agg gag ctc aca gcg tgt att ttc ccc aca gag tct gaa gca aga 912 Lys Arg Glu Leu Thr Ala Cys Ile Phe Pro Thr Glu Ser Glu Ala Arg 290 295 300 gca tga 918 Ala 305 23 329 PRT Artificial Description of Artificial Sequence fused protein composed of HitTag-PTD-MITF A-type N-terminal region (1-305) 23 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly Met Gln Ser Glu Ser Gly Ile Val 20 25 30 Ala Asp Phe Glu Val Gly Glu Glu Phe His Glu Glu Pro Lys Thr Tyr 35 40 45 Tyr Glu Leu Lys Ser Gln Pro Leu Lys Ser Ser Ser Ser Ala Glu His 50 55 60 Ser Gly Ala Ser Lys Pro Pro Leu Ser Ser Ser Thr Met Thr Ser Arg 65 70 75 80 Ile Leu Leu Arg Gln Gln Leu Met Arg Glu Gln Met Gln Glu Gln Glu 85 90 95 Arg Arg Glu Gln Gln Gln Lys Leu Gln Ala Ala Gln Phe Met Gln Gln 100 105 110 Arg Val Ala Val Ser Gln Thr Pro Ala Ile Asn Val Ser Val Pro Thr 115 120 125 Thr Leu Pro Ser Ala Thr Gln Val Pro Met Glu Val Leu Lys Val Gln 130 135 140 Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala Gln Arg 145 150 155 160 His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys His Ala 165 170 175 Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp His Ala 180 185 190 Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met Ala Met 195 200 205 Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu 210 215 220 Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr His Ser 225 230 235 240 Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile Ile Ser 245 250 255 Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp Pro Ala 260 265 270 Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile Asp Leu 275 280 285 Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser 290 295 300 Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Ala Cys Ile 305 310 315 320 Phe Pro Thr Glu Ser Glu Ala Arg Ala 325 24 990 DNA Artificial Description of Artificial Sequence DNA coding the fused protein composed of HisTag-PTD-MITF A-type N-terminal region (1-305) 24 atg ggg ggt tct cat cat cat cat cat cat ggt ggt tat ggc agg aag 48 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 aag cgg aga cag cga cga aga ggt atg cag tcc gaa tcg gga atc gtg 96 Lys Arg Arg Gln Arg Arg Arg Gly Met Gln Ser Glu Ser Gly Ile Val 20 25 30 gcg gat ttc gaa gtc ggg gag gag ttt cac gaa gaa ccc aaa acc tat 144 Ala Asp Phe Glu Val Gly Glu Glu Phe His Glu Glu Pro Lys Thr Tyr 35 40 45 tac gaa ctc aaa agt caa cct ctg aag agc agc agt tct gca gag cat 192 Tyr Glu Leu Lys Ser Gln Pro Leu Lys Ser Ser Ser Ser Ala Glu His 50 55 60 tct ggg gcc tcc aag cct ccg tta agc tcc tcc act atg aca tca cgc 240 Ser Gly Ala Ser Lys Pro Pro Leu Ser Ser Ser Thr Met Thr Ser Arg 65 70 75 80 atc ttg cta cgc cag caa ctc atg cgt gag cag atg cag gag cag gag 288 Ile Leu Leu Arg Gln Gln Leu Met Arg Glu Gln Met Gln Glu Gln Glu 85 90 95 cgc agg gag cag cag cag aag ctg cag gca gcc cag ttc atg caa cag 336 Arg Arg Glu Gln Gln Gln Lys Leu Gln Ala Ala Gln Phe Met Gln Gln 100 105 110 aga gtg gcc gtg agt cag aca cca gcc ata aac gtc agc gtg ccc acc 384 Arg Val Ala Val Ser Gln Thr Pro Ala Ile Asn Val Ser Val Pro Thr 115 120 125 acc ctt ccc tct gcc acc cag gtg ccg atg gaa gtc ctt aag gtg cag 432 Thr Leu Pro Ser Ala Thr Gln Val Pro Met Glu Val Leu Lys Val Gln 130 135 140 acc cac ctg gaa aac ccc acc aag tac cac ata cag caa gct cag agg 480 Thr His Leu Glu Asn Pro Thr Lys Tyr His Ile Gln Gln Ala Gln Arg 145 150 155 160 cac cag gta aag cag tac ctt tct acc act tta gca aat aaa cat gcc 528 His Gln Val Lys Gln Tyr Leu Ser Thr Thr Leu Ala Asn Lys His Ala 165 170 175 agc caa gtc ctg agc tca cca tgt cca aac cag cct ggc gac cat gcc 576 Ser Gln Val Leu Ser Ser Pro Cys Pro Asn Gln Pro Gly Asp His Ala 180 185 190 atg cca cca gtg ccg ggg agc agc gca ccc aac agc cct atg gct atg 624 Met Pro Pro Val Pro Gly Ser Ser Ala Pro Asn Ser Pro Met Ala Met 195 200 205 ctc act ctt aac tcc aac tgt gaa aaa gag gca ttt tat aag ttt gag 672 Leu Thr Leu Asn Ser Asn Cys Glu Lys Glu Ala Phe Tyr Lys Phe Glu 210 215 220 gag cag agc agg gca gag agt gag tgc cca ggt atg aac acg cac tct 720 Glu Gln Ser Arg Ala Glu Ser Glu Cys Pro Gly Met Asn Thr His Ser 225 230 235 240 cga gcg tcg tgc atg cag atg gat gat gta att gat gac atc atc agc 768 Arg Ala Ser Cys Met Gln Met Asp Asp Val Ile Asp Asp Ile Ile Ser 245 250 255 ctg gaa tca agt tat aat gaa gaa att ttg ggc ttg atg gat ccg gcc 816 Leu Glu Ser Ser Tyr Asn Glu Glu Ile Leu Gly Leu Met Asp Pro Ala 260 265 270 ttg caa atg gca aat acg tta ccc gtc tct gga aac ttg atc gac ctc 864 Leu Gln Met Ala Asn Thr Leu Pro Val Ser Gly Asn Leu Ile Asp Leu 275 280 285 tac agc aac cag ggc ctg cca ccg cca ggc ctt acc atc agc aac tcc 912 Tyr Ser Asn Gln Gly Leu Pro Pro Pro Gly Leu Thr Ile Ser Asn Ser 290 295 300 tgt cca gcc aac ctt ccc aac ata aaa agg gag ctc aca gcg tgt att 960 Cys Pro Ala Asn Leu Pro Asn Ile Lys Arg Glu Leu Thr Ala Cys Ile 305 310 315 320 ttc ccc aca gag tct gaa gca aga gca tga 990 Phe Pro Thr Glu Ser Glu Ala Arg Ala 325 25 31 DNA Artificial Description of Artificial Sequence primer HLH F 25 gagacgaaga ggtatgttgg ctaaagagag g 31 26 30 DNA Artificial Description of Artificial Sequence primer pTD3-MITFa 26 cgccgcggaa tgcagtccga atcgggaatc 30 27 35 DNA Artificial Description of Artificial Sequence primer MITFR-N 27 gaattcacta tgctcttgct tcagactctg tgggg 35 28 75 DNA Artificial Description of Artificial Sequence DNA coding for fused proteins comprised of HisTag-linker-PTD-linker-MITF 28 atg ggg ggt tct cat cat cat cat cat cat ggt ggt tat ggc agg aag 48 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 aag cgg aga cag cga cga aga ggt atg 75 Lys Arg Arg Gln Arg Arg Arg Gly Met 20 25 29 25 PRT Artificial Description of Artificial Sequence DNA coding for fused proteins comprised of HisTag-linker-PTD-linker-MITF 29 Met Gly Gly Ser His His His His His His Gly Gly Tyr Gly Arg Lys 1 5 10 15 Lys Arg Arg Gln Arg Arg Arg Gly Met 20 25 

1: A cell death inducer for mast cells having a fusion protein comprising a PTD (protein transduction domain) and an MITF (microphthalmia-associated transcripution factor) variant as an active ingredient. 2: The cell death inducer according to claim 1, wherein the fusion protein comprises a His Tag, a PTD, and an MITF variant. 3: The cell death inducer according to claim 1, wherein the MITF variant is an MITF mi variant, wh variant, HLH (basic-helix-loop-helix/leucine zipper) fragment, or A-type N-terminal region (1-305) fragment, and the PTD is a TAT-derived peptide. 4: The cell death inducer according to claim 1, wherein the fusion protein has the amino acid sequence shown by SEQ ID NO: 11, 13, 15, or 23 in the Sequence Listing. 5: The cell death inducer according to claim 4, resulting from substituting, deleting, inserting, or adding one or a plurality of amino acids in the fusion protein, and having a substantially equivalent activity. 6: The cell death inducer according to claim 1, wherein the active ingredient is obtained by treating the fusion protein with a denaturing agent (chaotropic agent) and subsequently removing said denaturing agent. 7: The cell death inducer according to claim 1, wherein the denaturing agent is urea, guanidine hydrochloride, or thiocyanate. 8: A preventive/therapeutic agent for a disease in which mast cells are implicated, having a fusion protein comprising a PTD and an MITF variant as an active ingredient. 9: The preventive/therapeutic agent according to claim 8, wherein the disease in which mast cells are implicated is an allergy, asthma, an autoimmune disease, pulmonary fibrosis, a carcinoma, mastocytoma, or mastocytosis. 10: The preventive/therapeutic agent according to claim 8, wherein the fusion protein comprises a His Tag, a PTD, and an MITF variant. 11: A fusion protein comprising a His Tag, a PTD, and an MITF variant, wherein the MITF variant is an MITF mi variant, wh variant, HLH fragment, or A-type N-terminal region (1-305) fragment, and the PTD is a TAT-derived peptide. 12: The fusion protein according to claim 11, wherein the fusion protein has the amino acid sequence shown by SEQ ID NO: 11, 13, 15, or 23 in the Sequence Listing. 13: The fusion protein according to claim 12, wherein the fusion protein is resulted from substituting, deleting, inserting, or adding one or a plurality of amino acids, and has a substantially equivalent activity. 14: A pharmaceutical composition comprising the fusion protein according to claim 11 and a pharmacologically acceptable carrier. 15: The pharmaceutical composition according to claim 14, wherein a concentration of the fusion protein is 0.1-100 μg/ml or 0.1-100 nM. 16: A preparation method for the fusion protein according to claim 11, comprising a step of producing the fusion protein using a genetic engineering technique. 17: The preparation method according to claim 16, further comprising the steps of denaturing agent treatment, Ni column treatment, and denaturing agent removal. 18: The preparation method according to claim 16, wherein the fusion protein is produced using an Escherichia coli-derived host expression system. 19: The preparation method according to claim 16, wherein the denaturing agent removal is performed by a step of anion exchange or dialysis. 20: The preparation method according to claim 16, wherein the denaturing agent is urea, guanidine hydrochloride, or thiocyanate. 21: The preparation method according to claim 16, wherein the concentration of denaturing agent, which is added during denaturing agent treatment, is 1-10 M. 22: A DNA coding for the fusion protein according to claim
 11. 23: The DNA according to claim 22, wherein the fusion protein has the amino acid sequence shown by SEQ ID NO: 11, 13, 15, or 23 in the Sequence Listing. 24: The DNA according to claim 23, wherein the fusion protein is resulted from substituting, deleting, inserting, or adding one or a plurality of amino acids, and has a substantially equivalent activity. 25: The DNA according to claim 22, wherein said DNA has the base sequence shown by SEQ ID NO: 12, 14, 16, or 24 in the Sequence Listing. 26: A method for inducing cell death in mast cells comprising administrating an effective dose of a fusion protein comprising a PTD and an MITF variant. 27: The induction method according to claim 26, wherein the administration is intravenous, subcutaneous, intramuscular, percutaneous, or tracheobronchial. 28: The induction method according to claim 26, wherein the fusion protein is administered at a dose of 10 μg-50 mg. 29: The induction method according to claim 26, wherein the fusion protein comprises a His Tag, a PTD, and an MITF variant. 30 (canceled) 31 (canceled) 